1. Field of the Invention
The invention relates to electro-optical sensing devices for detecting the presence or concentration of an analyte in a liquid or gaseous medium. More particularly, the invention relates to an electro-optical sensing device having a signal channel responsive to the presence of an analyte in a liquid or gaseous medium and a reference channel that is not responsive to the presence of the analyte in the medium.
2. Discussion of the Background Art
U.S. Pat. No. 5,517,313, the disclosure of which is incorporated herein by reference, describes an electro-optical sensing device that detects the presence and amount of an analyte using fluorescence of an indicator molecule. Broadly speaking, in the context of the field of the present invention, indicator molecules are molecules having one or more optical characteristics affected by the local presence of an analyte. In one embodiment of the device according to U.S. Pat. No. 5,517,313, a light source is located at least partially within a layer of material containing indicator molecules that fluoresce when illuminated by the light source. Alternatively, the light source is located at least partially within a wave guide layer such that light emitted by the source strikes and causes the indicator molecules to fluoresce. A high-pass filter allows fluorescent light emitted by the indicator molecules to reach a photosensitive element while filtering out scattered light from the light source.
The fluorescence of the indicator molecules employed in the device described in U.S. Pat. No. 5,517,313 is modulated, i.e., attenuated or enhanced, by the local presence of an analyte. For example, the orange-red fluorescence of the complex tris(4,7-diphenyl-1,10-phenanthroline)ruthenium(II) perchlorate is quenched by the local presence of oxygen. Therefore, this complex can be used advantageously as the indicator molecule in an oxygen sensor. Indicator molecules whose fluorescence properties are affected by various other analytes are known as well.
In the sensing device described in U.S. Pat. No. 5,517,313, the material containing the indicator molecules is permeable to the analyte. Thus, the analyte can diffuse into the material from the surrounding test medium, thereby affecting the fluorescence of the indicator molecules. The light source, indicator molecule-containing material, high-pass filter, and photodetector are configured such that fluorescent light emitted by the indicator molecules impacts the photodetector such that an electrical signal is generated that is indicative of the concentration of the analyte in the surrounding medium.
In order to make accurate measurements based on a single variable, such as analyte concentration, the design of the sensing device must isolate the effects of analyte concentration from all other variables that may influence operation of the device. One way to do this is to measure all other influential variables using specific transducers, and assuming the relationship is well defined and predictable, compensate for these factors mathematically. Importantly, this method requires a means of measuring the influential variable specifically, and a detailed mathematical model describing and predicting its behavior over time.
In the example of the indicator molecule Ruthenium tris-biphenylphenanthroline, for example, the relationship between indicator fluorescence and oxygen concentration is described by the Stern-Volmer equation:I/IO=1+KSV[pO2]where I/IO is an intensity ratio, pO2 is oxygen concentration, and KSV is the Stern-Volmer constant. If the output from a sensor constructed using this type of indicator were recorded from within an environment which isolated all variables except oxygen concentration, we should see a plot as shown in FIG. 1. One can employ the Stern-Volmer plot as shown to measure an intensity in the presence and absence of oxygen (the analyte) and then find the corresponding oxygen concentration from the x-axis using the plot as a calibration curve.
In reality, however, KSV is a function of temperature, and in most practical applications, the temperature of the test medium can be expected to change substantially. The temperature can also change very rapidly. To account for the temperature sensitivity of KSV, the Stern-Volmer relationship can be represented by a series of curves, as shown in FIG. 2, corresponding to different temperatures.
Introduction of a second variable such as temperature thus makes measurement of the first variable (i.e., analyte concentration) much more difficult. It is necessary to know the temperature accurately in order to know which of the Stern-Volmer plots to use for finding the correct oxygen concentration from a measured intensity value.
An example of another influential variable is signal drift. Drift is less predictable than temperature because a multitude of known, and often, unknown, factors are causative. One of many such drift examples is illustrated by photo-oxidation in the case of the indicator Ruthenium tris-biphenylphenanthroline. Photo-oxidation, or photo-bleaching, is a well described degradation which occurs when a (typically photochemical) reaction occurs between the indicator and ambient oxygen. (In the case of the indicator Ruthenium tris-biphenylphenanthroline, the photodegradation occurs due to singlet oxygen.) This degradation reaction results in the covalent and permanent alteration of the indicator molecular structure. Once oxidized, the indicator loses it known performance characteristics and its sensitivity to the intended analyte. If a variable drift component due to photo-oxidation is superimposed over the previous temperature dependent variable shown in FIG. 2, the result is a complex plot of the type shown in FIG. 3. FIG. 3 shows what a Stern-Volmer calibration plot looks like under the influence of only three variables—changing oxygen concentration, changing temperature, and changing amplitude as a result of ongoing drift due to photo-oxidative degradation. It is not possible to know which of the plots to use for finding oxygen concentration without knowledge of the temperature and the amount of degradation experienced by the sensor.
Yet another example of error that may be introduced is from variable excitation light levels. Since the excitation light source directly “pumps” the fluorescence detected from the indicator, any fluctuation or degradation in the source light will directly be introduced as error into the calibration. Light source drift can be caused by transient changes in the sensor power supply or due to simple operational life degradation in the light source itself. Some means of correcting for this drift is necessary to making an accurate analyte measurement from sensor-supplied data.
From the above, it will be appreciated that, in order to design an electro-optical sensing device for the purpose of measuring a single analyte, some means of correcting for kinetic, molecular stability, and system influences which would otherwise introduce error into the measurement is required. These influential factors can become complex and are highly interdependent. For example, an increase in temperature will also tend to increase the rate of degradation due to photo-oxidation. There are many more known and often unknown influential factors than the three examples described. The result is a very complex and difficult to understand series of interdependent variables that directly affect the accuracy of a measurement by an electro-optical sensing device.
One method of correcting for the matrix of potential variables is to construct a reference channel that is responsive to all variables except the presence of the analyte in the external environment. The output from the reference channel may then be used to cancel the effect of such variables on the sensing channel, for example by taking the ratio of the sensing and reference channel outputs. In the absence of any change in the amount of analyte in the external environment, this ratio should remain constant over time so that, if the ratio is plotted, the result would be a flat line. This ensures that any change in the output ratio is due entirely to any change in the amount of analyte in the external environment.
Several examples of using a reference channel in this manner during analyte detection are known in the art. For example, U.S. Pat. No. 3,612,866, the entire disclosure of which is incorporated herein by reference, describes a fluorescent oxygen sensor having a reference channel containing the same indicator chemistry as the measuring channel, except that the reference channel is coated with varnish to render it impermeable to oxygen. U.S. patent application Ser. No. 09/383,148, filed Aug. 26, 1999 and entitled “Optical-Based Sensing Devices” (which issued as U.S. Pat. No. 6,330,464 on Dec. 11, 2001), the disclosure of which is incorporated herein by reference, discloses another fluorescent oxygen sensing device having a reference channel that starts out with the same base chemistry as the sensing or signal channel but is further processed to block oxygen diffusion, for example by coating the reference channel with a material that is impermeable to oxygen.
This approach, however, may induce other differences between the channels that cannot be canceled by taking the ratio of the outputs. For example, the output from the reference channel may be increased or decreased due to reflectivity of the coating material. If the gain stages for each channel are designed the same, one could be running at substantially higher levels than the other due to differences in reflectivity. In addition, ambient light that might be present in the external environment at the same wavelength as the fluorescent emission would probably not be picked-up by the reference channel and, thus, would probably not be canceled.
Yet another difference may stem from the fact that surface chemical properties of the coating create the dominant properties of the reference channel whereas the chemical properties of the indicator material or matrix create the dominant properties of the sensing channel. Susceptibility to dust and condensation, chemical compatibility, and wear, would be expected to create other differences.
It is also expected that mechanical micro-thermal influences driven by surface turbulence would be different in a reference channel that has been “blanketed” and protected. Moreover, specific absorption or diffusion characteristics may be different. The rate of photo-oxidation would be expected to be dramatically different as the light scattering or absorbing influence from the coating may intensify the excitation flux on the indicator molecule. This would result in different rates of photo-bleaching thereby removing a key benefit sought from use of the reference channel.
Importantly, the inherent solubility of the analyte within the coating material will establish the concentration as seen by the reference channel. For example, if the analyte is oxygen, the inherent solubility of the coating material for oxygen will be the oxygen level maintained at the interface between the coating material and the top surface of the reference channel. Assuming this solubility results in an equilibrium concentration much less than the concentration of oxygen in air, then the reference channel will “see” a relatively anoxic environment. It will therefore perform as if it were in an anoxic environment. If the indicator molecule were a Ru complex as mentioned above, then the fluorescence of the reference channel will be much greater than the signal channel at sensor baseline because of the inverse relationship between oxygen quenching and fluorescence intensity. Further, because there is less oxygen in equilibrium with the reference channel on average in this example, the rate of photo-oxidation (beyond the previously described light scatter influence) will be reduced by the ratio of oxygen in the coating versus oxygen in air. Any chemical reaction with alteration of, or inclusion of, the chemical components of the coating material within the indicator matrix upon initial application will alter the performance characteristics relative to the signal channel.
FIG. 4 illustrates an optical sensing device 10 of the same general type as described in U.S. Pat. No. 6,330,464 having an excitation source 12 in the form of an LED mounted on a substrate 14 within a housing 16, a pair of indicator membranes 18A and 18B mounted over openings 20A and 20B formed in the housing, and a pair of photosensitive elements 22A and 22B on opposite sides of the LED. The indicator membranes have the same base chemistry, however, the indicator membrane shown on the right in FIG. 4 is coated with a material 24 that is impermeable to oxygen in an attempt to form a reference channel.
FIG. 5 is a graph of actual test results for a sensing device of the type shown in FIG. 4 illustrating significantly different responses for the signal and reference channels over an extended period of time during which the sensing device is exposed to ambient air having a constant oxygen concentration. It can be seen that the ratio of the signal and reference channel outputs is not a flat line as desired, but an increasing function that makes interpretation of the results complex.
In addition to the foregoing, there are other methods of using a reference during analyte detection. For example, U.S. Pat. Nos. 4,861,727 and 5,190,729, the entire disclosures of which are incorporated herein by reference, describe oxygen sensors employing two different lanthanide-based indicator chemistries that emit at two different wavelengths, a terbium-based indicator being quenched by oxygen and a europium-based indicator being largely unaffected by oxygen. U.S. Pat. No. 5,094,959, the entire disclosure of which is also incorporated herein by reference, describes an oxygen sensor in which a single indicator molecule is irradiated at a certain wavelength and the fluorescence emitted by the molecule is measured over two different emission spectra having two different sensitivities to oxygen. Specifically, the emission spectra which is less sensitive to oxygen is used as a reference to ratio the two emission intensities. U.S. Pat. Nos. 5,462,880 and 5,728,422, the entire disclosures of which are also incorporated herein by reference, describe a radiometric fluorescence oxygen sensing method employing a reference molecule that is substantially unaffected by oxygen and has a photodecomposition rate similar to the indicator molecule. Additionally, Muller, B., et al., ANALYST, Vol. 121, pp. 339–343 (March 1996), the entire disclosure of which is incorporated herein by reference, describes a fluorescence sensor for dissolved CO2, in which a blue LED light source is directed through a fiber optic coupler to an indicator channel and to a separate reference photodetector which detects changes in the LED light intensity.
In addition, U.S. Pat. No. 4,580,059, the entire disclosure of which is incorporated herein by reference, describes a fluorescent-based sensor containing a reference light measuring cell for measuring changes in the intensity of the excitation light source—see, e.g., column 10, lines 1, et seq. Furthermore, U.S. Pat. No. 4,617,277, the entire disclosure of which is also incorporated herein by reference, describes an absorbance-based sensor for carbon monoxide, in which a reference element reflects light from a source to a reference photocell to determine when a measuring element needs replacement due to irreversible color change.
There remains a need in the art for an electro-optical sensing device with a reference channel that responds in essentially the same manner as the measuring channel to all environmental and systemic factors except the presence of an analyte of interest in the external environment.